Bacterial dipeptidyl-peptidase (DPP) 7 liberates a dipeptide with a preference for aliphatic and aromatic penultimate residues from the N-terminus. Although synthetic substrates are useful for activity measurements, those currently used are problematic, because they are more efficiently degraded by DPP5. We here aimed to develop a potent and specific substrate and found that the kcat/Km value for Phe-Met-methylcoumaryl-7-amide (MCA) (41.40 ± 0.83 μM−1 s−1) was highest compared to Met-Leu-, Leu-Leu-, and Phe-Leu-MCA (1.06–3.77 μM−1 s−1). Its hydrolyzing activity was abrogated in a Porphyromonas gingivalis dpp7-knockout strain. Conclusively, we propose Phe-Met-MCA as an ideal synthetic substrate for DPP7.